Aim 1. To investigate biologic consequences of BDNF activation of TrkB on chemoresistance. Inhibition of AKT pathway in neuroblastoma inhibits tumor cell growth in vitro and in vivo. Our previous studies have identified activation of the PI3Kinase/Akt/GSK3 pathway mediates resistance to chemotherapy in neuroblastoma cells. Moreover our genetic and pharmacologic studies indicated that activation of AKT alone attenuated the effects of chemotherapy in neuroblastoma cells. Since activated AKT is more highly expressed in tumors of Neuroblastoma patients with a poor prognosis, we screened a number of inhibitors of the AKT that could enhance the efficacy of chemotherapy in our pre-clinical in vitro models. We focused on the AKT inhibitor, Perifosine, because it is Phase I/II trials in adult cancers, with a toxicity profile that is controllable with systemic therapy. Perifosine was tested in a number of our pre-clinical in vitro models as well as our in vivo murine heterotypic and orthotopic xenograft models. Perifosine inhibits activation of AKT and more impressively inhibits tumor cell growth in all 4 cell line models tested in vitro and in vivo. In the AS neuroblastoma model system there was complete tumor regression. In the least sensitive model, the addition of chemotherapy with Perifosine caused dramatic tumor regressions. These studies provide proof of principle that targeting Akt alone will inhibit neuroblastoma cell growth and will synergize with cytotoxics such as etoposide. Specific Aim 2. To investigate biologic consequences of BDNF activation of TrkB on metastasis and angiogenesis Our previous studies have identified that activation of TrkB stimulated increases in HIF-1alpha and production of VEGF in Neuroblastoma. Moreover we have found that increases in TrkB stimulate increased invasiveness. In order to model this in vivo additional neuroblastoma Tet-regulated TrkB expression models have been generated so that the cell lines contain the spectrum of genetic alterations found in poor prognosis neuroblastoma tumors. Moreover, we have developed in vitro models that contain more physiologically relevant levels of O2 and evaluated how hypoxia affects the sensitivity of neuroblastoma cells to a number of biologic response modifiers. Since TrkA expression is associated with good prognosis and elevated TrkA levels had been previously identified to suppress VEGF levels we initiated a study to assess whether NGF activation of TrkA altered VEGF expression. Our evidence supported previous study that elevated TrkA in the absence of NGF suppressed VEGF. However we found that NGF activation in TrkA expressing NB cells stimulated VEGF production via activation of HIF-1a. However, in assessing normal dorsal root ganglion TrkA expressing neurons from mice we determined that NGF activation of TrkA induces HIF-1a and increases in VEGF production. Specific Aim 3. Develop in vivo model systems to test targeted agents to the BDNF/TrkB signal transduction pathway Elevated levels of TrkB are associated with poor prognosis in patients with Neuroblastoma. Functionally we have found that activation of TrkB stimulates neuroblastoma migration and enhances survival under nutrient deprivation and when exposed to chemotherapy in in vitro models. We have developed an in vivo model in which the level of TrkB expression in tumor cells is regulated by tetracycline. We have found that mice whose tumors have increased levels of TrkB are less sensitive to low dose chemotherapy than mice whose tumors have lower levels of TrkB. Current studies are aimed at evaluating whether inhibition of the TrkB/PI-3Kinase/Akt pathway re-sensitizes tumor cells to chemotherapy. The Akt inhibitor Perifosine was evaluated and found to re-sensitize NB cells expressing high levels of TrkB to etoposide. These studies were first performed in "in vitro" assays and similar results were also seen in our murine model of TrkB induced chemoresistance. This indicates that targeting Akt in NB tumors should sensitize them to chemotherapy. This may be an effective strategy for relapsed neuroblastoma tumors.